The goal is to understand the evolution of microRNAs (miRNAs), the coevolution between miRNAs and their targets, and the role miRNA-target interactions play in the expression divergence between closely related species of Drosophila. miRNAs are small regulatory RNAs, which target mRNA transcripts to repress their expression. Aim 1: The evolution of miRNA genes - Emergence of new miRNA genes and changes in either the sequences of mature miRs or their expression will be studied by extensive sequencing of small RNAs from six tissues in five species of Drosophila. Aim 2: The effects of miRNAs on the evolution of gene expression (I. miRNAs) - First, four conservative miRNAs will be used to transform D. melanogaster and D. simulans. Second, four pairs of evolving miRNAs will be used to transform D. melanogaster. The effects of the transgenes on expression will be assayed by microarrays. Aim 3: The effects of miRNAs on the evolution of gene expression (II. targets) - We will verify and calibrate the results of D2 by selecting the 3'UTR of 15 target genes for reporter assay. Aim 4: The fitness effects of the coevolution between miRNAs and targets - If miRNAs and targets are co-adapted, then miRNAs and target transcripts that have been evolving separately would likely show fitness incompatibilities. This hypothesis, in the frame work of Muller-Dobzhansky model, will be tested. Project Narrative: Micro-RNAs are a large class of regulators of the activities of other genes. They are known to function in animal/plant development, cancer, complex traits and genetic diseases. A survey of the scope of microRNA repertoire and their rise and fall through evolution will be useful for understanding their roles in regulating the normal and aberrant phenotypes in human populations.